Experiments are described whose purpose is to advance understanding of the mechanism of inhibition of adenylate cyclase by muscarinic cholinergic agonists. A membrane preparation from rat heart will be utilized to determine the point in the adenylate cyclase regulatory cycle at which muscarinic receptors interact to regulate adenylate cyclase. The nucleotide dependence of muscarinic receptor-mediated inhibition of adenylate cyclase will be compared to the role of nucleotides in beta-adrenergic receptor-mediated activation. The relationship of the effects of nucleotides and ions on muscarinic receptor binding to the role of these factors in muscarinic receptor-mediated inhibition of adenylate cyclase will be assessed. Chemical manipulations for differentially affecting the inhibitory component(s) of the adenylate cyclase system will be developed. Cultured heart cells will be utilized to examine the regulation of cyclic AMP levels by muscarinic receptors in intact cells and to assess the possibility of an agonist-induced uncoupling of muscarinic receptors from a nucleotide binding site. The possibility of an agonist-induced association of muscarinic receptors with a guanine nucleotide binding protein will be studied using a gel exclusion chromatography. Reconstitution experiments will be carried out to determine unambiguously whether muscarinic receptors and beta-adrenergic receptors interact with a common guanine nucleotide binding protein.